Neuropeptides are important in transducing changes through their interaction with receptors in the extracellular milieu. Endopeptidase EC 3.4.24.15 (EP24.15) is a soluble metallopeptidase with broad expression in the brain that is involved in neuropeptide metabolism. Enzymes like EP24.15 either: inactivate peptides, convert an inert precursor to an active moiety, or in some instances, convert on bioactive peptide into another with different agonist properties. While the soluble, cytosolic forms of these enzymes have been characterized in purified form, including EP24.15, the properties of the secreted enzyme, much less the secretory mechanisms for the enzymes, have not been extensively studied. Thus, it is important to characterize the enzyme with respect to targeting to the extracellular milieu and the factors modulating enzyme activity extracellularly. This will be accomplished in three Specific Aims. Specific Aim 1: How is EP 24.15 enzyme targeted to the extracellular milieu? Aim 1a. Are there different forms of the EP24.15 derived from the same or different MRNAs? Are the membrane-bound and free ribosome mRNA forms different? What is the structure of the EP24.15 encoding gene? Aim 1b. Determine the secretory pathways targeting the secreted and plasma membrane forms of EP24.15 Which secretory pathway for EP24.15 is subserved by the individual EP24.15 mRNAs? Does mutation of the phosphorylation site and/or cysteinyl mutants change EP24.15 release? Specific Aim 2: Characterize the association between EP24.15 and the plasma membrane or extracellular matrix (ECM). Aim 2a. What is the biochemical differences between plasma membrane and soluble EP24.15? Is the plasma membrane/media form of EP24.15 proteolytically processed? How is EP24.15 associated with the plasma membrane? Aim 2b. Which specific ECM components bind EP24.15 and which EP24.15 are involved in the interaction(s)? Aim 2c. What are the effects of phosphorylation and redox status on EP24.15's association with the extracellular environment? Is the amino terminal domain of EP24.15 important in this association? Specific Aim 3: Determine whether EP24.15 interactions with the cell surface of ECM can modulate EP24.15 activity. Aim 3a. Are the changes in EP24.15 redox-dependent multimerization between different extracellular compartments? Do the mutations which abolish thiol activation of the recomb. enzyme similarly affect the extracellular pool of EP24.15? Aim 3b. Does phosphorylation alter the activity/partition of EP24.15 in the extracellular environment? Determine which form of EP24.15 and the residue on the EP24.15 protein for phosphorylation in vivo.